Abstract

Fibroblast growth factor (FGF)21 is an endocrine hormone that is expressed in multiple tissues and functions physiologically to maintain energy homeostasis. FGF21 is being pursued as a therapeutic target for diabetes and obesity because of its rapid and potent effects on improving insulin sensitivity. However, whether FGF21 enhances insulin sensitivity under physiologic conditions remains unclear. Here, we show that liver-derived FGF21 enters the circulation during fasting but also remains present and functional during the early stage of refeeding. After a prolonged fast, FGF21 acts as an insulin sensitizer to overcome the peripheral insulin resistance induced by fasting, thereby maximizing glucose uptake. Likewise, FGF21 is produced from the liver during overfeeding and mitigates peripheral insulin resistance. DIO FGF21 liver-specific knockout, but not FGF21 adipose-specific knockout, mice have increased insulin resistance and decreased brown adipose tissue–mediated glucose disposal. These data are compatible with the concept that FGF21 functions physiologically as an insulin sensitizer under conditions of acute refeeding and overfeeding.

Introduction

Fibroblast growth factor (FGF)21 is an endocrine hormone that signals through a cell-surface receptor complex composed of a classic FGF receptor, FGFR1c, and the FGF coreceptor, β-klotho (1). FGF21 is expressed in several tissues including liver, white adipose tissue (WAT), brown adipose tissue (BAT), and pancreas and has multiple proposed physiological functions (2), some of which remain controversial (3). Pharmacologically, FGF21 is a potent insulin sensitizer that improves metabolic dysfunction in a number of obese animal models and humans (4,5). A single pharmacological dose of FGF21 to obese mice can decrease plasma glucose levels by ∼40–50% within 1 h, an effect lasting up to 6 h (6). Additionally, extended administration of FGF21 to obese rodents and primates significantly increases energy expenditure and weight loss (7–9). Although incompletely understood, adipose tissue is particularly important for the pharmacological actions of FGF21 as the hormone’s acute glucose-lowering effect is lost in mice lacking either β–klotho (10) or FGFR1c in adipose tissue (11). Here, we show that circulating FGF21 levels are completely derived from the liver during prolonged fasting and diet-induced obesity (DIO). Interestingly, loss of FGF21 in the liver, but not adipose tissue, impairs insulin-mediated glucose uptake during refeeding and overfeeding. These data show that FGF21 functions as an insulin sensitizer under specific physiologic conditions.

Research Design and Methods

Animals

FGF21 knockout (12), albumin-Cre (13), adiponectin-Cre (14,15), and FLP-transgenic mice (16) have previously been described. FGF21fl/fl mice were generated by crossing the FGF21neo-loxP/+ mice (12) with FLP-transgenic mice to remove the neo cassette. Subsequent FGF21fl/+ mice were backcrossed seven generations to C57Bl/6 mice. FGF21fl/+ mice were then crossed together to generate FGF21fl/fl mice. FGF21fl/fl mice were then crossed to albumin-Cre or adiponectin-Cre transgenic mice (C57Bl/6J background) to generate liver-specific FGF21 knockout (FGF21fl/fl;Albumin-Cre) or adipose-specific FGF21 knockout (FGF21fl/fl;Adiponectin-Cre) mice, respectively. Mice were maintained on a chow (2920X; Teklad Global Diets, Harlan Laboratories) or high-fat diet (HFD; Research Diets [D12492i]). Twenty-four hour fasting experiments were performed from 9 a.m. to 9 a.m., and refeeding was performed via oral gavage as previously described (17). Plasma glucose, insulin, triglycerides, nonesterified fatty acid (NEFA), and β-hydroxybutyrate were determined as previously described (12,17). For measurement of plasma glucagon, plasma was mixed with aprotinin and then snap-frozen in liquid nitrogen and stored at −80°C until assayed by the Vanderbilt Hormone Assay & Analytical Services Core. Plasma FGF21 was measured using an FGF21 ELISA assay (BioVendor). All procedures and use of mice were approved by the Institutional Animal Care and Use Committee of the University of Iowa.

In Vivo Glucose Uptake Assays

Six-week-old male wild-type (WT) and FGF21 liver-specific knockout mice (FGF21 LivKO) were placed on an HFD for 6 weeks. After an overnight fast, tail blood was collected from all mice (time = 0). All mice were then injected with 8–10 µCi [3H]2-deoxyglucose i.p. in a 20% glucose solution and tail blood was collected over the course of 60 min (15 min, 30 min, and 60 min). At the end of the time course, all mice were killed by decapitation and tissues immediately dissected, flash frozen in liquid nitrogen, and placed at −80°C until analysis. Plasma radioactivity (18) and determination of tissue-specific uptake of [3H]2-deoxyglucose was performed as previously described (19,20).

Isolation of Primary Brown and White Adipocytes

Primary brown and white preadipocytes were isolated from the intrascapular brown fat and white fat depots, respectively, of 4-day-old C57Bl6 pups as previously described (21).

In Vitro Glucose Uptake

Primary adipocytes were treated with one of the following conditions prepared in glucose-free media: vehicle, insulin (100 nmol/L), FGF21 (1 µg/mL), or insulin combined with FGF21. Recombinant FGF21 generation and purification have previously been described (12). Adipocytes were treated for 60 min, followed by in vitro glucose uptake assays performed as previously described (7).

Data Analysis

Gene expression analyses were performed as previously described (12). Statistical comparison of two groups was determined using Student t test.

Results and Discussion

Plasma FGF21 Levels Are Derived From the Liver and Regulate Fasting and Refeeding Responses

To examine the physiological conditions regulating FGF21 levels, we profiled plasma FGF21 protein levels during fed, fasted, or refed conditions and compared them with circulating insulin levels. Plasma FGF21 levels were increased with 24 h fasting and remained elevated 15 min after refeeding (Supplementary Fig. 1A), when insulin levels were also elevated (Supplementary Fig. 1B). The increased circulating FGF21 concentrations observed after refeeding are likely residual levels produced during fasting, as circulating FGF21 levels return to those of the fed state by 1 h (Supplementary Fig. 1A), consistent with the half-life of FGF21 in mice being <30 min (9). Notably, the time period immediately after refeeding is a unique physiological condition in which both FGF21 and insulin are circulating.

To determine which tissues contribute to circulating FGF21, we generated FGF21 LivKO mice by crossing FGF21fl/fl mice with albumin-Cre transgenic mice. Fed, fasted, or refed WT and FGF21 LivKO mice were analyzed. While Fgf21 mRNA was induced in the livers of fasted and refed WT mice, Fgf21 mRNA expression was completely abolished in the livers (Fig. 1A), but not epididymal WAT (eWAT) (Fig. 1B), of FGF21 LivKO mice. Plasma FGF21 was induced during fasting and refeeding in WT mice but was abolished in FGF21 LivKO mice (Fig. 1C). There was no difference in the level of fed plasma glucose between groups. However, fasted FGF21 LivKO mice exhibited a slight, but significant, decrease in plasma glucose (Fig. 1D). Importantly, refed FGF21 LivKO mice had significantly elevated plasma glucose compared with WT littermates (Fig. 1D) despite having similar plasma insulin (Fig. 1E) and adiponectin (Fig. 1F) levels. FGF21 LivKO mice displayed an elevation in their glucose excursion curves compared with WT littermates when subjected to a glucose tolerance test (GTT) (Fig. 1G), despite having insulin levels similar to control (data not shown). Together, these data show that the function of FGF21 extends beyond fasting into the early refeeding response to enhance insulin-stimulated glucose uptake.

FGF21 Is Induced in Liver by HFD Feeding to Drive Glucose Disposal Into BAT In Vivo

To determine which tissue(s) have reduced glucose disposal and may be responsible for the DIO FGF21 LivKO phenotype, we analyzed tissue-specific glucose uptake in WT and FGF21 LivKO mice fed an HFD for 6 weeks. This was accomplished by injecting mice with labeled [3H]2-deoxyglucose in a 20% glucose bolus to assess glucose uptake during a physiological insulin response. FGF21 LivKO mice on HFD for 6 weeks also exhibited an increased glucose excursion curve compared with WT controls (Fig. 3A). Notably, this increased glucose excursion in the FGF21 LivKO mice fed HFD for 6 weeks occurred despite having normal plasma insulin levels (Fig. 3B), unlike FGF21 LivKO mice fed HFD for 10 weeks (Fig. 2G). Interestingly, FGF21 LivKO mice had a lower rate of glucose uptake in BAT, but not in eWAT, subcutaneous WAT, skeletal muscle, or heart (Fig. 3C). We conclude that FGF21 regulates glycemia at least in part by effecting glucose disposal into BAT in DIO mice. FGF21 has also been shown to reduce hepatic glucose production during obesity (rev. in 2), so multiple mechanisms may be responsible for FGF21-mediated effects on glucose homeostasis. FGF21 enhances glucose disposal under specific physiologic conditions, and a limitation of this study is that only glucose metabolism was examined. Thus, we cannot rule out that other metabolic pathways are regulated by FGF21 under these or other physiological conditions.

We next examined whether FGF21 can act directly on brown adipocytes to stimulate glucose uptake. Primary brown and white preadipocytes were isolated, differentiated, and then treated with vehicle, insulin, FGF21, or both insulin and FGF21 for 1 h. While extended treatment (24 h) of white adipocytes with FGF21 stimulates glucose uptake independent of insulin (7), 1-h treatment with FGF21 alone did not stimulate glucose uptake in either brown (Fig. 3D) or white (Fig. 3E) adipocytes. Insulin alone, however, was able to significantly increase glucose uptake in brown (Fig. 3D) and white (Fig. 3E) adipocytes. Interestingly, cotreatment of insulin with FGF21 synergistically increased glucose uptake in primary brown adipocytes (Fig. 3D) but not white adipocytes (Fig. 3E). These data demonstrate that FGF21 can signal directly to brown adipocytes to enhance insulin-stimulated glucose uptake and that loss of circulating FGF21 impairs glucose uptake in BAT of DIO mice. These results, however, do not exclude the possibility that FGF21 may also regulate BAT activity through indirect mechanisms (e.g., sympathetic nerve activity) in vivo. Therefore, just as FGF21 functions early during refeeding to mitigate physiological peripheral insulin resistance, FGF21 may also function during overfeeding to overcome diet-induced insulin resistance.

In summary, our data are compatible with FGF21 functioning beyond the fasting/starvation response to enhance insulin action during refeeding and overfeeding. Thus, FGF21 acts as an insulin sensitizer under physiologic conditions, which may explain its acute pharmacological actions. FGF21 may promote glucose uptake into adipose during refeeding to maximize energy replenishment and during overfeeding to protect against lipotoxicity. Interestingly, the ability of FGF21 to enhance insulin action may explain why pharmacological administration of FGF21 increases insulin sensitivity in obese mice, where circulating insulin levels are already elevated, but not in lean mice. Understanding the mechanisms regulating FGF21-mediated enhancement of insulin sensitivity may provide important insight into new treatments for metabolic disease.

Article Information

Funding. This work was supported by an American Diabetes Association Junior Faculty award (7-13-JF-49 to M.J.P.), an Edward Mallinckrodt, Jr. Foundation grant (to M.J.P.), and a University of Iowa Carver Trust Medical Research Initiative grant (to M.J.P.). This work also received generous research support from the Fraternal Order of Eagles Diabetes Research Center (to M.J.P.), National Institutes of Health (NIH) grants R01-DK-067158 (to D.J.M. and S.A.K.) and R01-DE-013686 (to M.M.), the Robert A. Welch Foundation (grant I-1275 to D.J.M. and grant I-1558 to S.A.K.), and the Howard Hughes Medical Institute (to D.J.M.). The Vanderbilt Hormone Assay & Analytical Services Core (assayed plasma glucagon levels) is supported by NIH grant DK-20593.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. K.R.M. designed the experiments, performed in vitro and in vivo radioactive glucose uptake assays, and wrote the manuscript. M.C.N. performed in vitro and in vivo radioactive glucose uptake assays, performed gene expression analyses, and reviewed the manuscript. M.K.A. and M.D.A. performed gene expression analyses and reviewed the manuscript. D.J.M., S.A.K., and M.M. generated critical reagents and reviewed the manuscript. M.J.P. designed the experiments and wrote the manuscript. M.J.P. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.